Zentrum für Molekulare Biologie Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany.
Nat Struct Mol Biol. 2013 Apr;20(4):525-31. doi: 10.1038/nsmb.2526. Epub 2013 Mar 17.
SUMOylation is an essential modification that regulates hundreds of proteins in eukaryotic cells. Owing to its dynamic nature and low steady-state levels, endogenous SUMOylation is challenging to detect. Here, we present a method that allows efficient enrichment and identification of endogenous targets of SUMO1 and the nearly identical SUMO2 and 3 (SUMO 2/3) from vertebrate cells and complex organ tissue. Using monoclonal antibodies for which we mapped the epitope, we enriched SUMOylated proteins by immunoprecipitation and peptide elution. We used this approach in combination with MS to identify SUMOylated proteins, which resulted in the first direct comparison of the endogenous SUMO1- and SUMO2/3-modified proteome in mammalian cells, to our knowledge. This protocol provides an affordable and feasible tool to investigate endogenous SUMOylation in primary cells, tissues and organs, and it will facilitate understanding of SUMO's role in physiology and disease.
SUMOylation 是一种重要的修饰方式，能够调节真核细胞中数百种蛋白质。由于其动态性质和低稳态水平，内源性 SUMOylation 难以检测。在这里，我们提出了一种从脊椎动物细胞和复杂组织中有效富集和鉴定 SUMO1 及其几乎相同的 SUMO2 和 3（SUMO 2/3）的内源性靶标的方法。我们使用针对其表位进行了作图的单克隆抗体，通过免疫沉淀和肽洗脱来富集 SUMO 化蛋白质。我们将这种方法与 MS 结合使用来鉴定 SUMO 化蛋白质，这是首次直接比较哺乳动物细胞中内源性 SUMO1 和 SUMO2/3 修饰的蛋白质组，据我们所知。该方案为研究原代细胞、组织和器官中的内源性 SUMOylation 提供了一种经济可行的工具，并将有助于了解 SUMO 在生理和疾病中的作用。
